Stabilization of benzidine-type indicators with various enhancers

ABSTRACT

An improved composition, device and method, whereby enhanced sensitivity is achieved in detecting test sample constituents such as glucose and blood. The improvement resides in the use of a broad range of compounds as enhancers for benzidine-type indicators. The enhancer tends to stabilize the blue colorform of such indicators.

This is a division, of application Ser. No. 93,431, filed Nov. 13, 1979,now U.S. Pat. No. 4,290,773.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of analysis of a test samplefor the presence of a constituent. More particularly, it relates to acomposition capable of producing a detectable response in the presenceof the constituent. Such a composition lends itself to the detection ofvarious reducing sugars, hydrogen peroxide, peroxidase, peroxidativelyactive substances, hypochlorite and other analytes.

The analysis of test samples for the presence of sugars finds utility inmany unrelated arts. Thus, the present invention pertains to suchdiverse arts as the brewing industry, biochemical research and medicaldiagnostics. In the brewing industry, for example, starch is convertedto sugars, such as maltose, prior to actual fermentation. The presenceof maltose is therefore carefully monitored to assure high yields fromthe grain starting material. Many biochemical systems require glucose incarefully controlled concentrations as their cellular energy source, andthe research of such systems necessitates that these concentrations becarefully monitored. The medical profession utilizes sugar analysis to agreat extent in diagnosing and controlling such diseases as diabetesmellitus, which manifests itself by abnormally high glucoseconcentrations in the blood and urine.

Likewise many analytical methods are presently available for detectingthe presence of peroxidatively active substances in samples such asurine, fecal suspensions, and gastrointestinal contents. Hemoglobin andits derivatives are typical of such "peroxidatively active" substancesbecause they behave in a manner similar to the behavior of the enzymeperoxidase. Such substances are also referred to herein aspseudoperoxidases. Peroxidatively active substances are enzyme-like inthat they catalyze the redox reaction between peroxides and benzidine,o-tolidine, 3,3',5,5'-tetramethylbenzidine, 2,7-diaminofluorene orsimilar benzidine-type indicator substances, thereby producing adetectable response such as a color change. Most methods for determiningthe presence of occult blood in test samples rely on thispseudoperoxidase activity.

Thus, the field of the present invention extends to a very diverseassortment of pursuits. It finds applicability wherever sugar analysisbecomes a matter of significance, be it in brewing, the food industry,scientific research or medicine. Moreover, it lends itself to a varietyof techniques for determining the presence of a peroxidase orpseudoperoxidase. In fact, the present invention finds utility in anyfield where its unique propensity to exhibit a detectable response isadaptable. Any system which can ultimately provide H₂ O₂ as a reactionproduct or which contains peroxidase or a pseudoperoxidase is suitablefor application of the present invention, as are other systems such asswimming pool water containing hypochlorite and other strongly oxidizingsystems.

2. Description of the Prior Art

The history of sugar analysis is perhaps most noteworthy because it hasseen dramatic change over the years, both in the basic chemistriesutilized and in its format. For the most part these analyses can becharacterized as oxidizing systems which, when reduced, initiatereaction conditions leading to a detectable response, such as a colorchange or change in wavelength of ultraviolet light absorbed orreflected by the system. Thus, reducing sugars will convert silver oxideto metallic silver, and, if a solution of the sugar is applied to apiece of filter paper impregnated with silver oxide, a black dotdevelops. F. Feigl, Chem. Ind., Vol. 57, p. 1161, London (1938).Similarly, o-dinitrobenzene and the 3,4- and 3,5-isomers ofdinitrophthalic acid give a sensitive color reaction (forming violetshades) when heated with reducing sugars in Na₂ CO₃. T. Momose, et al.,Chem. Pharm. Bull. Tokyo, Vol. 12, p. 14 (1964); F. Feigl, Spot Tests inOrganic Analysis, 7th Edition, pp. 338-339, Elsevier Publ. Co., New York(1966).

But as early as 1849 it was known that reducing sugars would cause analkaline solution of CuSO₄ to precipitate the yellow to redCopper(I)oxide (oxyhydrate). H. Fehling, Ann., Vol. 72 (1849). See alsoB. Herstein, J. Am. Chem. Soc., Vol. 32, p. 779 (1910). This earlymilestone, known as the Fehling test, lent impetus to the development ofa far more sensitive test which utilized silver oxide in ammonia, theso-called Tollens reagent, which reacts readily with reducing agents toproduce a black precipitate of metallic silver, often forming a mirroron the inside walls of glass reaction vessels. B. Tollens, Ber., Vol.14, p. 1950 (1881); Vol. 15, p. 1635, 1828 (1882).

Because of the relatively high incidence of diabetes mellitus and itsaccompanying serious clinical consequences, high interest from thebiological and medical professions arose in new techniques for analyzingglucose levels in urine and serum. This keen interest led to thedevelopment of several procedures which deviate dramatically from theirsolution chemistry forbears. These utilize sophisticated biochemicalsystems which can be incorporated into dry, dip-and-read devices, usedin solution or suspension techniques, or in conjunction withspectrophotometers and other hardware.

Of these new techniques, the present invention lends itself especiallywell to an enzymatic system wherein the analyte, for instance glucose,is a substrate for a particular enzyme, the reaction products beingcapable of eliciting a detectable response from a family of indicatorcompounds known loosely in the art as "benzidine-type indicators." Thesewill be more carefully defined, infra, but for the present suffice it tosay these compounds can undergo color changes in the presence ofhydrogen peroxide and the enzyme peroxidase. The glucose/glucose oxidasesystem exemplifies the prior art, wherein glucose is oxidized togluconic acid with the concomitant formation of H₂ O₂ in accordancewith: ##STR1##

It is the concomitant formation of hydrogen peroxide which facilitatesthe subsequent, indicator-related steps leading to observable colorformation or other detectable response. Thus a benzidine-type indicatorresponds in the presence of hydrogen peroxide and peroxidase by changingits light absorptive capability.

In practice, this technology is presently utilized for glucose analysisin the form of dip-and-read reagent strips such as those marketed by theAmes Division of Miles Laboratories, Inc. under the trademark CLINISTIX®and others. Broadly, these comprise a plastic strip, at one end of whichis mounted an absorbent paper portion impregnated with the appropriateenzymes, indicator compound and buffering agents as the principal activeingredients. They are used by dipping the reagent-bearing end into thetest sample, removing it and comparing any color formed in the paperwith a standard color chart calibrated to various glucoseconcentrations.

Several patents have issued which are deemed pertinent to the presentinvention with respect to its application to glucose analysis. U.S. Pat.No. 2,848,308, issued to Alfred H. Free, disclosed and claimed the basicenzyme chemistry whereby glucose oxidase, peroxidase and abenzidine-type indicator are used in a reagent strip to determineglucose in urine or other bodily fluid. U.S. Pat. No. 3,753,863, issuedto Speck discloses the use of lower alkane polyols to "stabilize"indicator solutions of the benzidine type. Finally, U.S. Pat. No.4,071,317, issued to Lam, discloses the stabilization of an occultblood-sensitive composition through the use of certain sulfone,sulfoxide and amide compounds as diluents during preparation of thecomposition. This latter composition comprises an organic hydroperoxidecompound, and an indicator compound such as of the benzidine type.

As in the case of sugar analysis, several methods for peroxidase orpseudoperoxidase analysis have evolved over the years which rely onenzyme-like catalysis of the oxidation of color-forming indicators inthe presence of hydrogen peroxide. Primarily these include wet chemicalprocedures and "dip-and-read" type reagent-bearing strips. Of theformer, a typical example is set forth in Richard M. Henry, et al.,Clinical Chemistry Principles and Techniques, Hagerstown, Maryland:Harper and Row (1974), pp. 1124-1125. This procedure involves the use ofglacial acetic acid (buffer), diphenylamine (indicator), and hydrogenperoxide. While such wet methods have proven analytical ability, theyare nevertheless fraught with obvious shortcomings, not the least ofwhich are poor reagent stability and inadequate sensitivity. Inherent tosuch reagent solutions is a decline in stability (ergo sensitivity) sorapid that fresh reagent solutions must be prepared after several daysof storage, a necessity resulting in both excessive time required ofanalytical personnel, and poor economy because of having to waste costlyreagents.

A second method for the determination of peroxidatively activesubstances, and the one presently preferred by most clinical assayistsand analysts, utilizes "dip-and-read" reagent strips. Typical of suchdevices are reagent strips manufactured by the Ames Division of MilesLaboratories, Inc. and sold under the name HEMASTIX®. These comprise, inessence, a porous paper matrix affixed to a plastic strip or handle. Thematrix is impregnated with a buffered mixture of an organichydroperoxide and o-tolidine. Upon immersion in a liquid containinghemoglobin, myoglobin, erythrocytes or other pseudoperoxidases, a bluecolor develops in the matrix, the intensity of which is proportional tothe concentration of the peroxidatively active substance in the sample.Thus, by comparing the color developed in the matrix to a standard colorchart, the assayist can determine, on a semi-quantative basis, theamount of unknown present in the sample.

The advantages of reagent strips over wet chemistry methods arepredominantly twofold: strips are easier to use because neither thepreparation of reagents nor the attendant apparatus is required; andgreater stability of reagents is afforded, resulting in greateraccuracy, sensitivity and economy.

But the inherent advantages of strips over wet chemistrynotwithstanding, the characteristics of stability and sensitivity are inneed of still further improvement. Whereas these properties in currentstate-of-the art strips for determining pseudoperoxidases, sugars andother analytes are greatly preferred over those of wet chemical methods,there would nevertheless accrue a great advance in the art if suchstrips could be made even more stable during storage and even moresensitive to their respective analytes.

At least three attempts at improving pseudoperoxidase-sensitive systemsare recorded in the prior art. A recitation in Chemical Abstracts Volume85, page 186 (1976) describes a two-dip method for preparing occultblood-sensitive reagent strips containing o-tolidine and phenylisopropylhydroperoxide. In this method, a solution was made of the indicator(o-tolidine.2HCl) and polyvinylpyrrolidone in ethanol. To this solutionwas added a small amount of surfactant and enough citrate buffer toprovide a pH of 3.7. Filter paper strips impregnated with ethylcellulose were dipped in this solution and dried. The thus-impregnatedfilter paper was subsequently dipped into a second solution containing1,4-diazabicyclo[2.2.2]octane, phenylisopropyl hydroperoxide andpolyvinylpyrrolidone dissolved in an ethanol-toluene mixture. The thrustof this experiment was to stabilize the peroxide and indicatorcombination through the use of the bicyclooctane derivative and thepolyvinylpyrrolidone.

A second such method is disclosed in U.S. Pat. No. 3,853,471. Thispatent teaches the use of phosphoric or phosphonic acid amides where thesubstituent amido groups are primarily N-morpholine radicals.

Besides these attempts, there also exists the disclosure of U.S. Pat.No. 3,252,762 wherein the organic hydroperoxide is physicallyencapsulated within a colloidal material such as gelatin. When such atest strip is utilized, the aqueous test sample dissolves the gelatincapsules, thereby freeing the hydroperoxide for further reaction withthe indicator in the presence of a peroxidatively active substance.

Each of these prior attempts was aimed at stabilizing the reagents sothat the potentially incompatible reactive ingredients (hydroperoxideand indicator) would not prematurely combine and thereby render the teststrips less sensitive. Hence, it can be said that the prior art methodswere not directed towards the combined objectives of simultaneouslyenhancing stability and sensitivity, but rather they attempted topreserve existing sensitivity by preventing reagent decomposition duringstorage.

Another prior art reference which is of interest to one considering thegeneral concepts discussed herein in U.S. Pat. No. 3,236,850. Thispatent is directed towards stabilizing organic hydroperoxides used ascatalysts and oxidizing agents. The patentees in this reference disclosethe use of primary, secondary, or tertiary amine salts with organicperoxides. This reference is in no way directed toward reagent teststrips, or other analytical techniques.

To summarize the state of the art prior to the present invention,sugar-sensitive chemistries began to appear on the analytical scene asearly as the middle of the 19th century with the advent of Fehling'ssolution and Tollens' reagent. Most of the "purely chemical" systemswhich have since emerged have been largely superseded by biochemicalsystems, particularly those which comprise a sugar oxidase, peroxidaseand a peroxide-sensitive indicator of the benzidine type. These latterindicator compounds have been said to be stabilized by the presence oflower alkyl polyols.

Pseudoperoxidase-sensitive chemistries were also utilized early on aswet chemistry techniques, having given way to dip-and-read techniquesinvolving an organic peroxide and an indicator, such as a benzidinederivative, impregnated in a carrier matrix. Attempts at stabilizingthese reagents have included (a) the concomitant use of bicyclooctaneand polyvinylpyrrolidone, (b) phosphoric or phosphonic acid amides, (c)physical separation of reagents using gelatin capsules, and (d) primary,secondary and tertiary amine salts.

Finally, a composition sensitive to the presence of occult blood inurine is taught to be stabilized if formulated in the presence ofcertain sulfone, sulfoxide and/or amide compounds. There is no teachingto applicants' knowledge, anywhere in the prior art suggesting thepresently disclosed and claimed composition and test device, or methodfor their use.

SUMMARY OF THE INVENTION

Briefly stated, the present invention relates to an improvedcomposition, test device and method for detecting the presence of aconstituent in a test sample. The composition is capable of producing adetectable response, such as a color change, in the presence of theconstituent; and comprises a benzidine-type indicator and an enhancercompound having the structure ##STR2## or mixtures thereof, wherein R islower alkyl, R' is H or lower alkyl, R" is lower alkylene, and X is OH,CN, NO₂, CHO, SO₃ R' or NRCOR', and wherein R--OH is a monoalkanol.

The present invention also includes a test device comprising a carriermatrix incorporated with the composition. The presence of the particularconstituent is determined by contacting the test sample with the deviceor composition, and observing any detectable response.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a ramification of the well-known"benzidine-type" indicator system. Benzidine and its derivatives havelong been used as chromogenic indicators in assays for such diverse testsample constituents as hypochlorite ion in swimming pool water, andglucose or occult blood in urine. Their ability to develop easilyrecognizable blue hues of varying intensities renders them capable ofboth qualitative and semi-quantitative utility. Since the presentinvention pertains to this indicator system on a broad scale, it isdeemed important to elucidate the types of compounds included within thescope of the term "benzidine-type" indicator, as well as many currentlyknown systems with which such indicators have been found to beefficacious.

Benzidine and its derivatives ("benzidine-type" indicators) are bestdefined in terms of the structure ##STR3## in which the R⁺ and R*substituents, same or different, can be hydrogen, lower alkyl (i.e.,alkyl having 1 to about 6 carbon atoms), lower alkyloxy (i.e., alkyloxyhaving 1 to about 6 carbon atoms), aryl or aryloxy. Moreover, the R*substituents can together form --CH₂)_(n) in which n is 1 or 2. Inaddition to the above characterizations of R⁺ and R* , it is understoodthat these groups, themselves, can be substituted such as with hydroxy,halogen, cyano, etc. Typical compounds embraced by the term"benzidine-type" indicator include benzidine, o-tolidine, o-dianisidine,3,3',5,5'-tetramethylbenzidine (hereafter "tetramethylbenzidine"),3,3',5,5'-tetra(alkyl)benzidine, the various N- and N'-substitutedbenzidines and others.

Although the mechanism of color formation from benzidine-type and otherindicators in the presence of certain analytes is not known to acertainty, it is known that two sequentially occurring colorformsresult: a first species which is blue in color, and a second which isbrown. Because the blue species tends to be transient, ultimatelymetamorphosing to the brown, it is necessary to look for the colorchange within a prescribed time period. Otherwise the true significanceof color change is lost, as subtle shades of blue-which are easilydistinguishable--give away to the less easily interpreted brown hues.The higher the analyte concentration in the test sample, the moreaggravated this problem becomes, due to the limiting effect on capacityto detect the higher ranges of analyte concentrations. Thus, it can beseen that it is highly advantageous to extend the duration of the bluespecies, thereby permitting greater differentiation betweenconcentrations, as well as providing higher and lower limits to thedetectable concentration ranges.

Moreover, because analytical tools such as reagent strips are not usedimmediately after manufacture, but are usually stored for relativelylong periods, and because too long a period between manufacture and usecan result in a loss in efficacy leading to false negative results,enhanced shelf life can be a marked asset: the better the shelf life,the more dependable the analytical results.

In addition to the benzidine-type indicator itself, the inventioncontemplates a myriad of reagent systems which, in the presence of aparticular analyte, promote the detectable indicator response, such as acolor appearance or change. Thus, if the present composition were to beemployed for hypochlorite determination, the indicator and enhancercomposition could be employed by themselves, no further reagents beingnecessary except, perhaps, a buffer.

For the determination of glucose in urine, on the other hand, a reagentsystem is employed comprising, in addition to the present composition,glucose oxidase, peroxidase and a suitable buffer. When such a system iscontacted with a urine sample containing glucose, the glucose oxidasecatalyzes the oxidation of glucose, yielding H₂ O₂ as a by-product. Inthe presence of peroxidase, the H₂ O₂ causes a color change orappearance in the benzidine-type indicator/enhancer composition. Thepurpose of the buffer, if included, is to optimize these reactions byproviding the most advantageous pH.

The determination of occult blood or other pseudoperoxidase, or ofperoxidase, requires still another reagent system in addition to thepresent composition: an organic hydroperoxide, such as cumenehydroperoxide, and, preferably, a suitable buffer. Thus, ifpseudoperoxidase is present in the test sample, the organicperoxide/pseudoperoxidase system will interact with the composition ofthe present invention to yield a color change enabling qualitative andsemi-quantitative pseudoperoxidase analysis.

The enhancer compound of the present invention has been found to promotesensitivity by permitting an observable color appearance at analytelevels lower than those possible with identical systems without theenhancer present. Similarly, the enhancer also permits analyte to besemi-quantitatively assesed at much higher concentrations. Thus, theentire range of analyte concentrations detectable with a particularreagent system and benzidine-type indicator may be expanded when theenhancer of the present invention is present in the formulation.

The compounds which are within the meaning of the term "enhancercompound" as used herein are indeed numerous. Moreover, these compoundstake on a myriad of seemingly unrelated types, including such chemicallydiverse compounds as phenol, acetonitrile, nitrobenzene, benzonitrile,ethylene carbonate, cyclohexanone, N-methylpyrrolidone, 4-picoline andpyrrole. Broadly, these compounds fall into the following genericcategories: ##STR4## wherein X is OH, CN, NO₂, CHO, SO₃ R' or NRCOR', Ris lower alkyl, R' is H or lower alkyl, R" is lower alkylene, and R--OHis a monoalkanol. By "lower alkyl" is meant a hydrocarbon radical havingfrom 1 to about 6 carbon atoms, including methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, tert-butyl, cyclobutyl, and all thevarious normal, branched and cyclic isomers of pentyl and hexyl.Moreover, R and R' can be substituted, such as with hydroxyl, halogen,cyano or other substituents. By "lower alkylene" is meant a divalentaliphatic radical having 1 to about 6 carbon atoms, including --CH₂ --,--CH₂ CH₂ --, and --CH₂ CH₂ CH₂ --.

The amount of enhancer used in conjunction with the benzidine-typeindicator is not critical, although an amount in the range of about 50to 800 or more mole percent based on the moles of indicator has beenfound to be effective in achieving the aforementioned enhanced indicatorsensitivity. Using this basis as a guideline, although not necessarily arequisite, the amounts of the present composition to be used with thevarious chemistries for the desired analytes can be easily determinedfrom the prior art, as well as from the Examples provided below.Moreover, given the present teachings, it would be a routine laboratoryexercise to determine optimum amounts.

The test device of the present invention comprises a carrier matrixincorporated with the indicator/enhancer composition. Moreover, it canbe additionally incorporated with any art-recognized or other reagentsystem useable with a benzidine-type indicator, such as the glucose- andpseudoperoxidase-sensitive systems discussed above.

The carrier matrix utilized in forming the test device can take on amultitude of forms. Thus, U.S. Pat. No. 3,846,247 teaches the use offelt, porous ceramic strips, and woven or matted glass fibers.Additionally, U.S. Pat. No. 3,552,928 teaches the use of wood sticks,cloth, sponge material, and argillaceous substances. The use ofsynthetic resin fleeces and glass fiber felts as a carrier matrix issuggested in British Pat. 1,369,139. Another British Pat. No. 1,349,623,proposes the use of light-permeable meshwork of thin filaments as acover for an underlying paper matrix. Polyamide fibers are taught inFrench Pat. No. 2,170,397. These suggestions notwithstanding, however,the material predominantly used in the art as a carrier matrix, and thatwhich is especially suitable for the present invention, is a bibulouspaper such as filter paper. It can thus be seen that there is a greatdeal of leeway in selecting an appropriate material for use as a carriermatrix, and the matrix can take on various physical forms. All of thesetypes are intended as being within the scope of the present invention.

Whichever is chosen, a web of carrier matrix material can beincorporated with the present composition in several ways. The web canbe drawn through a solution or suspension of the indicator and enhancerin an appropriate solvent such as water, methanol, benzene, cyclohexane,etc. After drying, the composition-laden matrix web can then be slitinto strips about 0.5 cm. wide and fastened to one edge of a strip ofplastic backing material measuring about 8 cm. in width. This can beachieved using a double-faced adhesive tape known as Double Stickavailable from the 3M Company. The backing material with the matrixstrip in place is then cut widthwise to form reagent strips measuring8×0.5 cm. having a 0.5 cm. square of reagent-laden carrier matrix at oneend, the other end serving as a handle.

Another way of incorporating a carrier matrix with the presentcomposition is by printing. U.S. Pat. No. 4,046,513 describes such atechnique whereby an ink comprising the composition is silk screenedonto a plastic carrier matrix. Still other ways, such as spraying thecomposition onto the matrix, are part of the prior art, and thus wouldbe within the ken of a person of ordinary skill in the art.

The following Examples are provided to further illustrate thecomposition and test device of the present invention, as well as themethod for their use. Included are the embodiments of the inventionpresently considered preferred, and which are presently deemed to be thebest mode of performance of the invention. Moreover, as can be seen fromthe foregoing discussion, the presently disclosed concepts are verybroad in scope; and the succeeding Examples should not be deemed asbeing in any way limiting.

EXAMPLES I-XI The Effects of Various Enhancers on o-Tolidine

Because o-tolidine is a benzidine-type indicator presently widelyemployed in commercial reagents for detecting glucose, occult blood,hypochlorous acid (hypochlorite), and other analytes, an experiment wasconducted to evaluate the effects of various enhancers of the presentinvention on the performance of that indicator. Specifically, a reagentcomposition comprising the indicator and one of various enhancers wascontacted with sodium hypochlorite solutions of increasingly higherconcentrations. The appearance of blue color, indicative of the presenceof hypochlorite, was observed as the amount of hypochlorite wasincreased, until the advent of brown color was observed. The amount ofhypochlorite required to induce the brown chromogen to the extent whereblue was not observable was then recorded as a function of the variousenhancers and a control system without any enhancer present.

A stock solution was prepared from the following ingredients, which wereadded in the order as listed.

    ______________________________________                                        Distilled water    184.0  milliliters (ml)                                    o-Tolidine . 2HCl  2.0    grams (g)                                           Acetone            116.0  ml                                                  Citrate buffer (pH 5)*                                                                           94.0   ml                                                  ______________________________________                                         *Prepared from 208 ml distilled water, 15.4 g citric acid (anhydrous) and     68 g Na.sub.3 citrate . 2H.sub.2 O.                                      

Eleven 19.7 ml aliquots of this stock solution were set aside. To ten ofthese was added 0.1 g of one of the enhancers listed in Table I.

Each aliquot thus prepared was used to impregnate a piece of Eaton &Dikeman 204 filter paper measuring 3.5 by 7 inches. The stock solutionwithout enhancer was used to impregnate another piece of filter paperfor use as a control. The impregnated papers were dried in a forced airoven at about 50° C. for about 10 minutes. Each dried paper was attachedto one side of a similar-sized sheet of double-faced adhesive tape (3MCompany, Double Stick Y-915). The resultant laminates were slit tomeasure 0.20×7 inches, and the remaining protective paper removed fromthe adhesive tape, whereupon the laminates were affixed along the wideredge of polystyrene sheets measuring 3.25×7 inches. Thepolystyrene/adhesive/impregnated paper composites were then slitperpendicular to their wider edge to provide plastic strip devices eachmeasuring 0.2×3.25 inches, having an impregnated paper portion measuring0.2 inches square at one end, the other end serving as a handle.

Each of the eleven sets of enhancer-containing devices, plus the controlset, was studied by brief immersion (1-2 seconds) of a device in ahypochlorite solution of known concentration. The hypochlorite solution(55 in all) were prepared in graduated concentrations ranging from 0.05gram/100 ml water (g%) to 2.75 g%, each succeeding solution being 0.05g% more concentrated than its predecessor. Strips of a particular setwere dipped in each of these solutions, and the appearance of color wasobserved after about 60 seconds. The highest concentration ofhypochlorite wherein blue color could be observed in a device after 60seconds was recorded. The data for the control and each enhancer testedis recorded in Table I.

                  TABLE I                                                         ______________________________________                                        Example No.                                                                              Enhancer        NaClO (g %)*                                       ______________________________________                                        I          Control         0.70                                               II         Phenol          0.85                                               III        Nitrobenzene    2.40                                               IV         Benzonitrile    2.20                                               V          Acetonitrile    1.40                                               VI         n-Hexanol       0.90                                               VII        Ethylene carbonate                                                                            2.45                                               VIII       Cyclohexanone   1.35                                               IX         Pyrrole         0.95                                               X          4-Picoline      1.10                                               XI         N-Methyl-2-pyrrolidone                                                                        2.15                                               ______________________________________                                         *Highest concentration wherein blue color was still observable 60 seconds     after dipping.                                                           

EXAMPLES XII--XXII The Effects of Various Enhancers on3,3',5,5'-Tetramethylbenzidine (TMB)

Experiments were conducted to assess the effects of the enhancers of thepresent invention on TMB.

The procedure followed was identical to that of Examples I-XI exceptthat 3.0 g TMB was substituted for o-tolidine in formulating the stocksolution. The results are tabulated in Table II.

                  TABLE II                                                        ______________________________________                                        Example No.                                                                              Enhancer        NaClO (g %)*                                       ______________________________________                                        XII        Control         0.65                                               XIII       Phenol          0.80                                               XIV        Nitrobenzene    2.50                                               XV         Benzonitrile    2.40                                               XVI        Acetonitrile    1.50                                               XVII       n-Hexanol       0.90                                               XVIII      Ethylene carbonate                                                                            2.60                                               XIX        Cyclohexanone   1.40                                               XX         Pyrrole         1.05                                               XXI        4-Picoline      1.30                                               XXII       N-Methyl-2-pyrrolidone                                                                        2.25                                               ______________________________________                                         *Highest concentration wherein blue color was still observable 60 seconds     after dipping.                                                           

The results of the experiments described in the Examples demonstrateconclusively the unexpected stabilization of the blue colorform of botho-tolidine and 3,3',5,5'-tetramethylbenzidine. With each of theseindicators, strong blue responses to increasing concentrations ofhypochlorite were in evidence in the presence of an enhancer of thepresent invention, whereas the controls (Examples I and XII) without anenhancer soon lost their blue hues, giving way to the secondary brownhues. In each case the presence of an enhancer yielded a more stableblue than did the controls.

What is claimed is:
 1. In a composition for detecting the presence of aconstituent in a test sample, said composition comprising abenzidine-type indicator, the improvement wherein said compositionadditionally comprises an enhancer compound having the structure R-CN,wherein R is lower alkyl.
 2. The composition of claim 1 wherein saidenhancer compound is acetonitrile.
 3. A test device for determining thepresence of a constituent in a test sample, said device comprising acarrier matrix incorporated with the composition of claim 1 or
 2. 4. Amethod for determining the presence of a constituent in a test sample,said method comprising contacting the said test sample with thecomposition of claim 1 or 2 and observing a detectable response.
 5. Amethod for determining the presence of a constituent in a test sample,said method comprising contacting said test sample with the device ofclaim 3 and observing a detectable response.